Galacto-oligosaccharide is a mixture of oligosaccharides, each constituted by galactose as a primary component and having a different bonding mode, and is generally manufactured by causing lactose to undergo transitional reaction using β-galactosidase. Galacto-oligosaccharide has been reported to have physiological benefits such as increasing the bifidobacteria in the intestines to regulate the enteric environment, and reducing mesenteric fat and thereby contributing to the prevention of adult diseases, and for some time has been found useful as a food for specified health use, functional food, or material thereof. If galacto-oligosaccharide is used as a component of food for specified health use, etc., quantitative analysis data must be displayed to guarantee its quality, compliance with standards, and use as a functional component, and accordingly there is a need for establishing a highly accurate quantification method.
One method to quantify galacto-oligosaccharides in beverages or food is to add β-galactosidase to act upon galacto-oligosaccharide, quantify the galactose produced as a result of enzyme decomposition, and calculate the quantity of oligosaccharide (Non-patent Literature 1). However, detecting the galactose being produced requires anion-exchange high-performance liquid chromatography fitted with an expensive pulse-type electrochemical detector, which keeps the general utility of this method low. Also, when the beverage or food contains lactose or other nutritional components, the galactose produced by enzyme treatment cannot be discriminated from the galactose separating from lactose, which consequently reduces the quantification accuracy of galacto-oligosaccharide.
Another method is available, whereby a characteristic component in galacto-oligosaccharide, such as 4′-galactosyl lactose (4′-GL; Galβ1-4Galβ1-4Glc), is quantified and the total quantity of galacto-oligosaccharide is calculated back from the quantity of 4′-GL. This method is particularly effective when the content of 4′-GL in the galacto-oligosaccharide to be used is known and this content is stable from one lot to another. For quantifying 4′-GL in a beverage or food sample, conventionally a method to separate other components in the beverage or food from 4′-GL by gel filtration chromatography based on molecular size is primarily used.
As mentioned above, however, galacto-oligosaccharide is often used as a functional material in nutrient-rich beverages and foods such as formulated powdered milk for infants and fermented milk products, and in many cases these beverages and foods contain sugar sources whose molecule is equivalent in size to that of 4′-GL, in which case these sugar sources cannot be separated from 4′-GL by gel filtration chromatography. For example, an attempt to separate and quantify 4′-GL in beverages or food containing maltotriose, which is a type of sugar contained in dextrin, by gel filtration chromatography would result in overlapping peaks from the two substances that have eluted from the column because maltotriose has the same molecular size as 4′-GL, thus affecting the quantification of 4′-GL or specifically the quantification of galacto-oligosaccharide.
Among the methods to detect oligosaccharide by reverse-phase chromatography, one that uses a C8 or C18 reverse-phase chromatography column is known (Patent Literature 1). However, an attempt to separate galacto-oligosaccharide in beverages or food using such column would fail to separate the galacto-oligosaccharide component from other components in the beverage or food.